Optogenetic Stimulation of Oxytocin Release

To confirm the endogenous release of oxytocin following the light stimulation of the PVN fibers, 5–6 weeks old females Wistar HAN rats (n = 3) received injections of the OT1-sensor virus into the vlPAG and the C1V1 virus into the PVN. Following a 2 weeks recovery period, rats were anesthetized by administering i.p. ketamine (Imalgene 300 mg/kg) and paxman (Rompun, 60 mg/kg). Transcardial perfusions were performed using an ice-cold, NMDG-based aCSF was used containing (in mM): NMDG (93), KCl (2.5), NaH₂PO₄ (1.25), NaHCO₃ (30), MgSO₄ (10), CaCl₂ (0.5), HEPES (20), D-Glucose (25), L-ascorbic acid (5), Thiourea (2), Sodium pyruvate (3), N-acetyl-L-cysteine (10) and Kynurenic acid (2.5). The pH was adjusted to 7.3–7.4 using HCl, after bubbling in a gas comprised of 95% O₂ and 5% CO₂. Rats were then decapitated, brains were removed and 350 μm thick coronal slices containing the vlPAG were obtained using a Leica VT1000s vibratome. Slices were placed in a recuperation chamber filled with normal aCSF at room temperature for at least 1 h. Normal aCSF was composed of (in mM): NaCl (124), KCl (2.5), NaH₂PO₄ (1.25), NaHCO₃ (26), MgSO₄ (2), CaCl₂ (2), D-Glucose (15), at pH 7.3−7.4 and continuously bubbled in 95% O₂–5% CO₂ gas. Osmolarity of all aCSF solutions were controlled to be between 290–310 mOsm. Finally, slices were transferred from the holding chamber to an immersion-recording chamber and superfused at a rate of 2 ml/min.